Simulated Microgravity Condition Alters the Gene Expression of some ECM and Adhesion Molecules in Adipose Derived Stem Cells

Adipose- derived stem cells (ADSCs) are widely used for tissue engineering and regenerative medicine. The beneficial effects of ADSCs on wound healing have already been reported. Remodeling of extracellular matrix (ECM) is the most important physiological event during wound healing. ECM is sensitive to mechanical stresses and the expression of its components can be therefore influenced. The aim of this study was to investigate the effect of simulated microgravity on gene expression of some ECM and adhesion molecules in human ADSCs. After isolation and characterization of ADSCs, cells were exposed to simulated microgravity for 1, 3 and 7 days. Real-time PCR, fluorescence immunocytochemistry, and MTT assay were performed to evaluate the alterations of integrin subunit beta 1 (ITGB1), collagen type 3 (ColIII), matrix metalloproteinase-1 (MMP1), CD44, fibrillin (FBN1), vimentin (VIM) genes, and ColIII protein levels as well as cells viability. Microgravity simulation increased the expression of ITGB1, ColIII, MMP1, and CD44 and declined the expression of FBN1 and VIM genes. ColIII protein levels also increased. There were no significant changes in the viability of cells cultured in microgravity. Since the high expression of ECM components is known as one of the fibroblast markers, our data suggest that pretreatment of ADSCs by simulated microgravity may increase their differentiation capacity towards fibroblastic cells. Microgravity had not adversely affected the viability of ADSCs, and it is likely to be used alone or in combination with biochemical inducers for cell manipulation.

cord blood, peripheral blood, dermis, amniotic fluid, and even in tumors (3)(4)(5)(6)(7). For the first time, adipose-derived stem cells (ADSCs) were introduced in 2001 as MSCs (8). ADSCs are propounded as a great source of MSCs that are easily achievable from adipose tissue via liposuction. In recent years, ADSCs are widely used for tissue engineering and regenerative medicine instead of using BMSCs because of their abundance in adipose tissue with minimal mortality, easy availability, and safe isolation (9). It also has been shown that ADSCs have therapeutic effects in wound healing and tissue repair studies (10). Today, impaired wound healing is challenging because of inadequate skin tissue in the site of injury. Therefore, stem cell biology provides the novel option for the cell therapy of wound repair.

Remodeling of the extracellular matrix (ECM)
is the most important physiological event during the wound healing process (11). ECM is the largest component of the normal skin, and its components play several key functions in wound healing process, such as providing support to lead cells into the injury area, and stimulating cells to proliferate and differentiate (11). Apart from the role of ECM in wound healing, it is also involved in a series of other cell activities including signal-transduction pathways, cell migration, and organization of cells into tissues and coordination of cell functions (12,13).
Studies have shown that the expression of ECM components can be influenced by mechanical stress (14). One of the most important mechanical factors that affect all types of life on earth is gravity. Previous reports have indicated that cultured cells like MSCs have responded to gravity (both microgravity and hypergravity), too (15,16).
Microgravity has been confirmed to affect growth and physiology of cell through impacting on intracellular signaling mechanisms, cell secretions, and gene expression (17,18). It has been shown that components of the cytoskeleton such as actin polymer are gravity sensitive and reorganized in microgravity condition. This can lead to changes in cell morphology and fate (19,20). Integrin is a mediated cell adhesion protein that connects the cytoskeleton to ECM (21). Vimentin, integrin, and CD44 are the proteins that play important roles in cell adhesion and ECM formation (22). With regard to ECM roles and the communication between ECM and cytoskeleton, it is expected that changes in mechanical forces have a significant effect on the ECM structure. Most researchers have used BMSCs to study the effect of simulated microgravity condition on the function and structure of stem cells (23,24). According to the variety in cell types and differences in their structure and functions, the effects of gravity on various cell types are different. Biowest, France) and used for the cell isolation.

Isolation of ADSCs
Cell isolation was done by enzymatic

Characterization of ADSCs
The isolated cells from passage 3 were

Functional characterization of ADSCs
ADSCs are multipotent stem cells that have the capacity to differentiate into osteoblasts, adipocytes, and chondrocytes (1). In this present work, to examine the multipotent potential of the

Fluorescence immunocytochemistry
To analyze the protein expression of collagen All experiments were replicated at least twice.

Isolation and culture of ADSCs
After 48 h of culture of SVF in cell culture flask, Fibroblast-like spindle shape cells were observed ( Figure 1). The adherent cells proliferated at a considerable rate and reached 70-80% confluence after 4 days. Both the shape and adhesion properties of the cells confirmed that the isolated cells were MSCs.

Flow cytometry analysis
As shown in figure 2, flow cytometric analysis of ADSCs showed that a higher percentage of ADSCs (>95%) expressed CD90, CD73, and CD105 (surface antigens of MSCs) but did not expressed CD34 and CD45 (hematopoietic markers) ( Figure 2G). These results showed that the isolated cells were MSCs.

Differentiation of isolated cells to adipocyte and osteoblast
To demonstrate the pluripotent capacity of ADSCs, they were differentiated into adipocyte and osteocyte.
Staining results by Oil Red O ( Figure 2B and 2C) and Alizarin Red S ( Figure 2E and 2F) confirmed that the isolated cells were differentiated well into adipocyte and osteoblast lineages (Figure 2).

Cell viability assay
The effect of simulated microgravity on ADSCs viability was determined using MTT assay.
As shown in Figure 3

Fluorescence immunocytochemistry
We analyzed the expression of collagen type III at the protein level using immunocytochemistry assay. Quantification of COLIII-positive area was performed using ImageJ 1.49v software. As seen in Figure 5, the expression of COLIII in simulated microgravity environment ( Figure 5C and 5D) is significantly higher than its expression in 1g condition ( Figure 5A and 5B) 5C, 5D, 5A and 5B, respectively; P<0.05). Also, it seems that simulated microgravity condition induced cells to generate aggregated and accumulated structure.  (19,20).

Discussion
Thus, simulated microgravity may be used as a novel methodology for manipulating cells along with other biochemical techniques (30,31).
In this study, we found that simulated Also, our data showed that ADSCs in simulated microgravity tended to form aggregated cell structures and grow in three-dimensional ways ( Figure 5D). This is one of the features of simulated microgravity impact on cells that was also reported by others (17). The potential of three-dimensional growth of cells under simulated microgravity condition offers an opportunity for tissue engineering without using scaffold for regenerative medicine.
We also found that simulated microgravity increased the expression of MMP1. MMP1, also known as fibroblast collagenase which breaks down the interstitial collagens including ColIII.
Surprisingly, we observed that simulated microgravity increased the expression of both